ARTICLE DOI: 10.1038/s41467-017-00505-8
OPEN
Hit-and-run programming of therapeutic cytoreagents using mRNA nanocarriers H.F. Moffett 1, M.E. Coon1, S. Radtke1, S.B. Stephan1, L. McKnight1, A. Lambert2, B.L. Stoddard2, H.P. Kiem1,3 & M.T. Stephan 1,3,4
Therapies based on immune cells have been applied for diseases ranging from cancer to diabetes. However, the viral and electroporation methods used to create cytoreagents are complex and expensive. Consequently, we develop targeted mRNA nanocarriers that are simply mixed with cells to reprogram them via transient expression. Here, we describe three examples to establish that the approach is simple and generalizable. First, we demonstrate that nanocarriers delivering mRNA encoding a genome-editing agent can efficiently knockout selected genes in anti-cancer T-cells. Second, we imprint a long-lived phenotype exhibiting improved antitumor activities into T-cells by transfecting them with mRNAs that encode a key transcription factor of memory formation. Third, we show how mRNA nanocarriers can program hematopoietic stem cells with improved self-renewal properties. The simplicity of the approach contrasts with the complex protocols currently used to program therapeutic cells, so our methods will likely facilitate manufacturing of cytoreagents.
1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. 2 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. 3 Department of Medicine, Division of Medical Oncology, University of Washington, Seattle, WA 98109, USA. 4 Department of Bioengineering and Molecular Engineering & Sciences Institute, University of Washington, Seattle, WA 98105, USA. Correspondence and requests for materials should be addressed to M.T.S. (email: [email protected])
NATURE COMMUNICATIONS | 8: 389
| DOI: 10.1038/s41467-017-00505-8 | www.nature.com/naturecommunications
1
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00505-8
T
herapeutic methods based on immune cells have experienced a substantial metamorphosis from interventions involving straightforward blood transfusions and bone marrow transplants into a nascent healthcare industry. Currently, over 500 companies are involved in the development and commercialization of cell-based therapeutic products1, and hematopoietic stem cell (HSC) transplants have evolved into the standard-of-care for treating leukemia and other bone and blood cancers (with over one million transplants performed worldwide to date2). But also, different sorts of cell therapy products are undergoing clinical evaluation for treating a variety of diseases, including tissue degeneration, chronic inflammation, autoimmunity, genetic disorders, cancer, and infections3–8. It has become possible to focus immune responses towards these diseases by genetically engineering T-cells to express targeted chimeric antigen receptors (CARs) or T cell receptors (TCRs), and this approach has presented positive clinical responses in cancer patients who have no other curative options9, 10. Thanks to a strong clinical presence, the expanding array of cell therapy products has catalyzed the field of cellular bioengineering with the goal of maximizing the therapeutic performance of these cytoreagents in patients11, 12. Some gene therapy applications require chronic expression systems that stably integrate the engineered transgene into the patient’s DNA. One example is the expression of cancer-specific receptor genes by T-cells, which converts them into ‘living drugs’ that can increase in number while they serially destroy tumor cells9, 10. Another is the introduction of gamma-globin genes into transplanted HSCs as a way to reverse beta thalassemia13. Despite the time and cost required for their production, as well as restrictions on the size and number of genes that they can package, viral vectors are currently the most effective means to stably express these transgenes14, 15. It is also possible to elicit phenotypic changes via transient expression of macromolecules, designed to accomplish “hit-andrun” genetic programming. In most of these kinds of applications, permanent expression of the therapeutic transgene is undesirable and potentially dangerous16. Examples include the use of
a
transcription factors to control cell differentiation17, 18, and the expression of sequence-specific nucleases to engineer genomes19. Although there is a growing number of applications where transient gene therapy could improve the curative potential of engineered cells, currently available methods (which, like the chronic expression methods described above, are mostly based on viral vectors) are complicated by the time and expense involved in the elaborate protocols required for their implementation20. Electroporation is an alternative transfection method, but physical permeabilization of plasma membranes compromises cell viability, which means these approaches are not suited for scale-up applications. Besides, like virus-based methods, electroporation cannot selectively transfect specific cell types from a heterogeneous pool, so it must be preceded by a cell purification process. Here, we describe a nanoreagent that, via a comparatively simple process, produces transient gene expression in cultured cells. We demonstrate that an appropriately designed messenger RNA (mRNA) nanocarrier can accomplish dose-controlled delivery of functional macromolecules to lymphocytes or HSCs simply by mixing the reagent with the cells in vitro (Fig. 1a). These nanoparticles (NPs) can be designed to target particular cell subtypes and, upon binding to them, stimulate receptormediated endocytosis, thereby introducing the synthetic mRNA they carry which the cells can now express. Because nuclear transport and transcription of the transgene are not required, this process is fast and efficient. Here, we illustrate in three examples how this new platform can be implemented to manufacture effective cell products for clinical use. In the first case, we used mRNA nanocarriers to edit the genome of T-cells and established that targeted delivery of mRNA encoding a rare-cleaving megaTAL nuclease21 into lymphocytes can efficiently disrupt their expression of T cell receptors. In the second application, we transiently expressed Foxo1 to reprogram the differentiation of effector cells into functionally competent memory cells22, 23. Our results demonstrate that engineered NPs can bias CAR-T-cells toward a central memory phenotype.
b Nanoparticle with synthetic mRNA cargo
Isolation of patient cells
Targeting ligand (Ab)
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–
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ARCA Kozak
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Selective binding and genetic programming
α globin 3′UTR PolyA 5-Methyl-CTP
Protein coding sequence Pseudo-UTP
U
C
A
(2) Proteins regulating cell fate, differentiation, viability of trafficking
Fig. 1 Creating mRNA nanoparticles to program therapeutic T-cells. a Schematic explaining how cultured T-cells can be programmed to express therapeutically relevant transgenes carried by polymeric nanoparticles (NPs). These particles are coated with ligands that target them to specific cell types, enabling them to introduce their mRNA cargoes and cause the targeted cells to express selected proteins (like transcription factors or genome-editing agents). b Design of targeted mRNA-carrying NPs. The inset shows a transmission electron micrograph of a representative NP; scale bar, 50 nm. Also depicted is the synthetic mRNA encapsulated in the NP, which is engineered to encode therapeutically relevant proteins 2
NATURE COMMUNICATIONS | 8: 389
| DOI: 10.1038/s41467-017-00505-8 | www.nature.com/naturecommunications
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00505-8
CD4
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op Un ar tra tic le nsfe -tr a ct El ns ed ec fe tro cte po d ra te d
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Fig. 2 mRNA nanoparticle transfection choreographs robust transgene expression by lymphocytes. a Primary T-cells were mixed with CD3-targeted polymeric nanoparticles (NPs) carrying Cy5-labeled mRNA. Confocal microscopy establishes that these particles are rapidly internalized from the cell surface. The images are representative of 15 randomly chosen fields. Scale bars, 2 μm. b Flow cytometry of preactivated PBMCs 24 h after incubation with CD3-targeted or isotype control antibody-targeted nanoparticles bearing eGFP-encoding mRNA. c Bar graph summarizing transfection efficiencies from three independent experiments conducted in duplicate. d, e Comparison of the effects electroporation and NP gene delivery have on cell expansion. Left panels show the workflow for transfection with NPs (top) and electroporation (bottom). Right panels show the -fold expansion of PBMC cultures from three independent donors treated with stimulatory beads on days 0 and 12. Matched cultures from each donor were not treated, or transfected using CD3/CD28-targeted NPs (d, right) or electroporation (e, right) on days 5 and 17. Every line represents one donor and each dot reflects the -fold T-cell expansion. Pairwise differences between groups were analyzed with the unpaired, two-tailed Student’s t test; n.s., non-significant; *, significant, n = 3). f Relative viability of NP-transfected and electroporated T-cells. Samples of 2 × 106 activated T-cells per condition were untreated, transfected with NPs, or electroporated. 18 h after treatment, cells were labeled with fluorescent dyes to assess viability. Results from three separate experiments conducted in duplicate are summarized in the bar graph shown in g. Statistical analysis between groups was performed using the unpaired, two-tailed Student’s t Test. *P < 0.0001 NATURE COMMUNICATIONS | 8: 389
| DOI: 10.1038/s41467-017-00505-8 | www.nature.com/naturecommunications
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00505-8
To assure that our transient gene delivery platform can easily be integrated into existing protocols for manufacturing other therapeutic cell types, we engineered our third example in HSCs. For many decades, these have been successfully used for treating hematological and immune diseases2. However, their limited number, especially when isolated from umbilical cord, prevents broader application of HSC-based therapies. Attempts to propagate these cells in vitro have generally failed, primarily because self-renewal and in vivo regenerative capacity are rapidly lost in culture24, 25. We used our nanotechnology platform to deliver transgenes into cultured HSCs, and demonstrated that transient expression of a key “stemness” regulator (Musashi-2 protein26) increases their capacity for self-renewal and maintains therapeutically desirable properties in the stem cells. The most significant benefit of our system is its simplicity in achieving genetic modifications of therapeutic cells at a clinical scale: all that is required is mixing the appropriate NP reagent with the cultured cells. Our approach patently contrasts with those currently used to transiently deliver genetic materials, which are less effective and involve many expensive and proprietary procedures that limit their availability. Beyond the T-cells and HSCs tested in our experiments, the technology described here could be adapted to improve the curative potentials of other cell types used in the clinic to treat disease (e.g., natural killer cells, regulatory T-cells, dendritic cells, or mesenchymal stem cells) without increasing handling time, risk, or complexity. Results Designing mRNA nanocarriers to choreograph gene expression. To create a reagent that can genetically modify primary T lymphocytes (which are notoriously refractory to non-viral transfection methods) simply by contact, we bioengineered polymeric NPs comprised of four functional components (Fig. 1b): (i) surface-anchored targeting ligands that selectively bind the NPs to T-cells and initiate rapid receptor-induced endocytosis to internalize them. In our experiments we used anti-CD3 and antiCD8 antibodies; (ii) a negatively charged coating that shields the NPs to minimize off-target binding by reducing the surface charge of the NPs. Because it is already widely used in drug delivery platforms, we selected polyglutamic acid (PGA) to accomplish this; (iii) a carrier matrix that condenses and protects the nucleic acids from enzymatic degradation while they are in the endosome, but releases them once the particles are transported into the cytoplasm, thereby enabling transcription of the encoded protein. For this, we used a biodegradable poly(β-amino ester) (PBAE) polymer formulation that has a half-life between 1 and 7 h in aqueous conditions; and (iv) nucleic acids that are encapsulated within the carrier and produce gene editing or transient expression of proteins that can permanently alter the phenotype of the T-cell. mRNA is an ideal platform for transient therapeutic protein expression, because it has no potential for genomic integration and does not require nuclear localization for expression. However, unmodified mRNA can activate intracellular toll-like receptors, limiting protein expression and leading to toxicity27. To improve the stability and reduce the immunogenic potential of the mRNA we deliver, we used synthetic versions that incorporate modified nucleotides. For example, substitution of uridine and cytidine with the engineered bases pseudouridine and 5-methyl-cytidine synergistically blocks identification by innate pattern recognition receptors and increases mRNA translation28. The NPs were manufactured utilizing a two-step, chargedriven self-assembly process. First, the synthetic mRNA was complexed with a positively-charged PBAE polymer, which condenses the mRNA into nano-sized complexes (Supplementary Fig. 1a, b). This step was followed by the addition of 4
antibody-functionalized PGA, which shields the positive charge of the PBAE-mRNA particles and confers lymphocyte-targeting. The resulting mRNA nanocarriers had a size of 109.6 ± SE/26.6 nm and an almost neutral surface charge (1.1 ± SE/5.3 mV zeta potential, Supplementary Fig. 1c). T-cell viability following mRNA nanocarrier transfection. Our goal is to streamline the manufacture of cell-based therapies, so we first tested whether simply adding targeted mRNA nanocarriers to an established culture of human lymphocytes is sufficient to choreograph robust transfection in them. We found that when CD3-targeted NPs carrying mRNA encoding a reporter (enhanced green fluorescent protein, eGFP) are incubated with these cells, they not only bind to them but also stimulate receptor-mediated endocytosis, providing entry for the genes they carry (Fig. 2a). Following a single NP application (NP:T cell ratio = 2 × 104:1), we routinely transfected >80% of these primary T-cells (Fig. 2b, c), with transgene expression observed as early as 5 h post-transfection (Supplementary Fig. 2a–c). Thus, this process is fast and efficient. Importantly, it was not necessary to prepare mRNA NPs freshly for each application—it was possible to lyophilize them before use with no change in properties or efficacy (Supplementary Fig. 2d). We also found that CD3targeted nanoparticles selectively bind T lymphocytes, as their interactions with off-target cells were low (Fig. 2b, c; Supplementary Fig. 3). T cell proliferation did not impair NP-uptake/ transfection, as (i) gene transfer into naive versus effector T-cells was comparable (Supplementary Fig. 4a, b), and (ii) addition of nanoparticles to stimulated T-cells at the peak of their expansion (day 17) yielded similar transfection relative to adding nanoparticles to freshly stimulated cells (day 5; Supplementary Fig. 4c). We next assessed the impact of targeted mRNA−carrying NPs on T-cell expansion. Because malignancies often progress quickly, it is important that engineered T-cells can be expanded to clinically relevant scales equally quickly. One widely used approach to multiply polyclonal lymphocytes is to incubate them with beads that are coated with antibodies against TCR/CD3 and co-stimulatory CD28 receptors. We found that even repeated transfections with CD3-targeted NPs did not interfere with T-cell expansion stimulated by these coated beads (Fig. 2d). These results contrast sharply with T-cell electroporation, which we tested side-by-side. Not only did electroporation add complex handling steps (Fig. 2e), it compromised viability of the lymphocytes (Fig. 2f, g) and reduced their yield by 60-fold (Fig. 2e, right panel). Nanoparticle methods integrate into CAR-T cell manufacture. To test our approach in a clinically relevant application, we incorporated NP-mediated mRNA transfection methods into the manufacture of leukemia-specific 19-41BBζ CAR T-cells (Fig. 3a). CD19-targeted receptors are the most investigated CAR-T cell product today, with nearly 30 ongoing clinical trials internationally29. Our ability to perform genome engineering offers the potential to improve the safety and efficacy of CAR-T-cells. For example, we can inhibit expression of endogenous TCRs to avoid graft-versus-host disease, or selectively delete immune checkpoint genes in these cells to strengthen their anti-cancer activity in the suppressive tumor milieu30, 31. Here, we tested the ability of NPs to deliver gene-editing agents by preparing particles carrying mRNA encoding megaTAL nuclease, which targets the constant region (TRAC) of the TCR alpha gene. Taking advantage of the flexibility offered by our NP formulation methods, we included mRNA for the DNA repair endonuclease TREX2 to improve knockout efficiency32, along with eGFP mRNA so we could track transfection. Control particles were loaded with eGFP
NATURE COMMUNICATIONS | 8: 389
| DOI: 10.1038/s41467-017-00505-8 | www.nature.com/naturecommunications
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00505-8
b
a
Day 0
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ne TC di R (g te -p en d) os om T it e TC ce ive ed R lls ite ne d) g T ativ ce e lls
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P = 0.8 (n.s.)
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Fig. 3 Nanoparticles can knockout T cell receptors in CAR-programmed lymphocytes. a Integration of nanoparticle (NP) transfection into normal manufacturing of CAR-T-cells. After stimulation with anti-CD3/CD28-coated beads (day 0), CD8-targeted mRNA NPs were introduced on days 1 and 2, then lentiviral transduction with a vector encoding the leukemia-specific 19-41BBz CAR was performed on day 3. We added either NPs carrying mRNAs encoding megaTAL nuclease plus eGFP, or control particles loaded with eGFP mRNA alone. b Flow cytometry of NP transfection efficiencies (based on eGFP signals) correlated with surface expression levels of TCRs (based on CD3 signals) by T-cells following NP treatments. c Summary plot showing editing efficiency as measured by loss of CD3 surface expression at day 14 (n = 6). d Surveyor assay confirming TCRα chain gene locus disruption. e Flow cytometry of lentiviral transduction in genome-edited versus control T-cells. f Bar graph showing mean viral transductions and SE of three independent experiments conducted in duplicate; n.s., not significant g, h Proliferation and cytolytic activity of TCR+ (FACS sorted TCR-positive, unedited 19-41BBz CAR-T-cells) and TCR- (FACS sorted TCR-negative, genome edited) 19-41BBz CAR-T-cells. To measure proliferation, T-cells were co-cultured on irradiated TM-LCL leukemia cells. Cytolytic assays were performed with CD19-expressing K562 target cells. i T cell IFN-γ release was measured with ELISA 48 h after stimulation on CD19+ TM-LCL leukemia cells or control LNCaP C4-2 prostate adenocarcinoma cells. Data from two experiments run in triplicate are shown
mRNA only. We found that, in contrast to eGFP-transfection (which did not impact TCR expression; Fig. 3b, top row), the addition of TCRα-megaTAL-carrying particles to the T-cell culture efficiently disrupted TCR expression by day 5, an effect that was maintained after loss of the mRNA by day 12 (Fig. 3b, bottom row). Average TCR knockout efficiency was 60.8% ( ± SE/17.7%; Fig. 3c), which corresponds with the percentage of NATURE COMMUNICATIONS | 8: 389
indel frequencies (a measure of targeting efficiency) determined using the Surveyor assay (Fig. 3d). Importantly, the presence of mRNA-carrying NPs did not affect virus-mediated gene transfer of the tumor-specific CAR, as we achieved equal transduction efficiencies with a lentiviral vector encoding 19-41BBζ CAR in NP-transfected and non-transfected T-cells (Fig. 3e, f). Following NP-mediated genome editing and lentiviral transduction,
| DOI: 10.1038/s41467-017-00505-8 | www.nature.com/naturecommunications
5
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00505-8
b
Relative to mode
Jurkat T
Relative Foxo13A mRNA
a T cells
MFI: 847 6,914
Isotype
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Control NPs (GFP+) Foxo13A-eGFP NPs (GFP+)
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CM signature (down) 0.0
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Enrichment score
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–0.35
0.0
Normalized enrichment score: 6.61
Normalized enrichment score: –8.77
FWER P-value:
OPEN
Hit-and-run programming of therapeutic cytoreagents using mRNA nanocarriers H.F. Moffett 1, M.E. Coon1, S. Radtke1, S.B. Stephan1, L. McKnight1, A. Lambert2, B.L. Stoddard2, H.P. Kiem1,3 & M.T. Stephan 1,3,4
Therapies based on immune cells have been applied for diseases ranging from cancer to diabetes. However, the viral and electroporation methods used to create cytoreagents are complex and expensive. Consequently, we develop targeted mRNA nanocarriers that are simply mixed with cells to reprogram them via transient expression. Here, we describe three examples to establish that the approach is simple and generalizable. First, we demonstrate that nanocarriers delivering mRNA encoding a genome-editing agent can efficiently knockout selected genes in anti-cancer T-cells. Second, we imprint a long-lived phenotype exhibiting improved antitumor activities into T-cells by transfecting them with mRNAs that encode a key transcription factor of memory formation. Third, we show how mRNA nanocarriers can program hematopoietic stem cells with improved self-renewal properties. The simplicity of the approach contrasts with the complex protocols currently used to program therapeutic cells, so our methods will likely facilitate manufacturing of cytoreagents.
1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. 2 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. 3 Department of Medicine, Division of Medical Oncology, University of Washington, Seattle, WA 98109, USA. 4 Department of Bioengineering and Molecular Engineering & Sciences Institute, University of Washington, Seattle, WA 98105, USA. Correspondence and requests for materials should be addressed to M.T.S. (email: [email protected])
NATURE COMMUNICATIONS | 8: 389
| DOI: 10.1038/s41467-017-00505-8 | www.nature.com/naturecommunications
1
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00505-8
T
herapeutic methods based on immune cells have experienced a substantial metamorphosis from interventions involving straightforward blood transfusions and bone marrow transplants into a nascent healthcare industry. Currently, over 500 companies are involved in the development and commercialization of cell-based therapeutic products1, and hematopoietic stem cell (HSC) transplants have evolved into the standard-of-care for treating leukemia and other bone and blood cancers (with over one million transplants performed worldwide to date2). But also, different sorts of cell therapy products are undergoing clinical evaluation for treating a variety of diseases, including tissue degeneration, chronic inflammation, autoimmunity, genetic disorders, cancer, and infections3–8. It has become possible to focus immune responses towards these diseases by genetically engineering T-cells to express targeted chimeric antigen receptors (CARs) or T cell receptors (TCRs), and this approach has presented positive clinical responses in cancer patients who have no other curative options9, 10. Thanks to a strong clinical presence, the expanding array of cell therapy products has catalyzed the field of cellular bioengineering with the goal of maximizing the therapeutic performance of these cytoreagents in patients11, 12. Some gene therapy applications require chronic expression systems that stably integrate the engineered transgene into the patient’s DNA. One example is the expression of cancer-specific receptor genes by T-cells, which converts them into ‘living drugs’ that can increase in number while they serially destroy tumor cells9, 10. Another is the introduction of gamma-globin genes into transplanted HSCs as a way to reverse beta thalassemia13. Despite the time and cost required for their production, as well as restrictions on the size and number of genes that they can package, viral vectors are currently the most effective means to stably express these transgenes14, 15. It is also possible to elicit phenotypic changes via transient expression of macromolecules, designed to accomplish “hit-andrun” genetic programming. In most of these kinds of applications, permanent expression of the therapeutic transgene is undesirable and potentially dangerous16. Examples include the use of
a
transcription factors to control cell differentiation17, 18, and the expression of sequence-specific nucleases to engineer genomes19. Although there is a growing number of applications where transient gene therapy could improve the curative potential of engineered cells, currently available methods (which, like the chronic expression methods described above, are mostly based on viral vectors) are complicated by the time and expense involved in the elaborate protocols required for their implementation20. Electroporation is an alternative transfection method, but physical permeabilization of plasma membranes compromises cell viability, which means these approaches are not suited for scale-up applications. Besides, like virus-based methods, electroporation cannot selectively transfect specific cell types from a heterogeneous pool, so it must be preceded by a cell purification process. Here, we describe a nanoreagent that, via a comparatively simple process, produces transient gene expression in cultured cells. We demonstrate that an appropriately designed messenger RNA (mRNA) nanocarrier can accomplish dose-controlled delivery of functional macromolecules to lymphocytes or HSCs simply by mixing the reagent with the cells in vitro (Fig. 1a). These nanoparticles (NPs) can be designed to target particular cell subtypes and, upon binding to them, stimulate receptormediated endocytosis, thereby introducing the synthetic mRNA they carry which the cells can now express. Because nuclear transport and transcription of the transgene are not required, this process is fast and efficient. Here, we illustrate in three examples how this new platform can be implemented to manufacture effective cell products for clinical use. In the first case, we used mRNA nanocarriers to edit the genome of T-cells and established that targeted delivery of mRNA encoding a rare-cleaving megaTAL nuclease21 into lymphocytes can efficiently disrupt their expression of T cell receptors. In the second application, we transiently expressed Foxo1 to reprogram the differentiation of effector cells into functionally competent memory cells22, 23. Our results demonstrate that engineered NPs can bias CAR-T-cells toward a central memory phenotype.
b Nanoparticle with synthetic mRNA cargo
Isolation of patient cells
Targeting ligand (Ab)
mRNA nanocarrier Targeting ligand (Ab) PGA Polymer Addition of nanoparticles to cell culture +
Reinfusion of programmed cells into patient
–
– PGA
–
–
Synthetic mRNA
ARCA Kozak
G
G
A
(1) Genome editing agents ± Genetic redirection using viral vectors encoding CARs or TCRs
Selective binding and genetic programming
α globin 3′UTR PolyA 5-Methyl-CTP
Protein coding sequence Pseudo-UTP
U
C
A
(2) Proteins regulating cell fate, differentiation, viability of trafficking
Fig. 1 Creating mRNA nanoparticles to program therapeutic T-cells. a Schematic explaining how cultured T-cells can be programmed to express therapeutically relevant transgenes carried by polymeric nanoparticles (NPs). These particles are coated with ligands that target them to specific cell types, enabling them to introduce their mRNA cargoes and cause the targeted cells to express selected proteins (like transcription factors or genome-editing agents). b Design of targeted mRNA-carrying NPs. The inset shows a transmission electron micrograph of a representative NP; scale bar, 50 nm. Also depicted is the synthetic mRNA encapsulated in the NP, which is engineered to encode therapeutically relevant proteins 2
NATURE COMMUNICATIONS | 8: 389
| DOI: 10.1038/s41467-017-00505-8 | www.nature.com/naturecommunications
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00505-8
CD4
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f Untransfected
Time (d) after initial stimulation
Electroporated T cell
Nanoparticletransfected
5 10 15 20 25
g Electroporated
1.05-fold
Viability (%) 89.9
87.1
41.8
80
1.7-fold *
60 40 20 0
op Un ar tra tic le nsfe -tr a ct El ns ed ec fe tro cte po d ra te d
Annexin V
N
an
7-AAD
100
Fig. 2 mRNA nanoparticle transfection choreographs robust transgene expression by lymphocytes. a Primary T-cells were mixed with CD3-targeted polymeric nanoparticles (NPs) carrying Cy5-labeled mRNA. Confocal microscopy establishes that these particles are rapidly internalized from the cell surface. The images are representative of 15 randomly chosen fields. Scale bars, 2 μm. b Flow cytometry of preactivated PBMCs 24 h after incubation with CD3-targeted or isotype control antibody-targeted nanoparticles bearing eGFP-encoding mRNA. c Bar graph summarizing transfection efficiencies from three independent experiments conducted in duplicate. d, e Comparison of the effects electroporation and NP gene delivery have on cell expansion. Left panels show the workflow for transfection with NPs (top) and electroporation (bottom). Right panels show the -fold expansion of PBMC cultures from three independent donors treated with stimulatory beads on days 0 and 12. Matched cultures from each donor were not treated, or transfected using CD3/CD28-targeted NPs (d, right) or electroporation (e, right) on days 5 and 17. Every line represents one donor and each dot reflects the -fold T-cell expansion. Pairwise differences between groups were analyzed with the unpaired, two-tailed Student’s t test; n.s., non-significant; *, significant, n = 3). f Relative viability of NP-transfected and electroporated T-cells. Samples of 2 × 106 activated T-cells per condition were untreated, transfected with NPs, or electroporated. 18 h after treatment, cells were labeled with fluorescent dyes to assess viability. Results from three separate experiments conducted in duplicate are summarized in the bar graph shown in g. Statistical analysis between groups was performed using the unpaired, two-tailed Student’s t Test. *P < 0.0001 NATURE COMMUNICATIONS | 8: 389
| DOI: 10.1038/s41467-017-00505-8 | www.nature.com/naturecommunications
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00505-8
To assure that our transient gene delivery platform can easily be integrated into existing protocols for manufacturing other therapeutic cell types, we engineered our third example in HSCs. For many decades, these have been successfully used for treating hematological and immune diseases2. However, their limited number, especially when isolated from umbilical cord, prevents broader application of HSC-based therapies. Attempts to propagate these cells in vitro have generally failed, primarily because self-renewal and in vivo regenerative capacity are rapidly lost in culture24, 25. We used our nanotechnology platform to deliver transgenes into cultured HSCs, and demonstrated that transient expression of a key “stemness” regulator (Musashi-2 protein26) increases their capacity for self-renewal and maintains therapeutically desirable properties in the stem cells. The most significant benefit of our system is its simplicity in achieving genetic modifications of therapeutic cells at a clinical scale: all that is required is mixing the appropriate NP reagent with the cultured cells. Our approach patently contrasts with those currently used to transiently deliver genetic materials, which are less effective and involve many expensive and proprietary procedures that limit their availability. Beyond the T-cells and HSCs tested in our experiments, the technology described here could be adapted to improve the curative potentials of other cell types used in the clinic to treat disease (e.g., natural killer cells, regulatory T-cells, dendritic cells, or mesenchymal stem cells) without increasing handling time, risk, or complexity. Results Designing mRNA nanocarriers to choreograph gene expression. To create a reagent that can genetically modify primary T lymphocytes (which are notoriously refractory to non-viral transfection methods) simply by contact, we bioengineered polymeric NPs comprised of four functional components (Fig. 1b): (i) surface-anchored targeting ligands that selectively bind the NPs to T-cells and initiate rapid receptor-induced endocytosis to internalize them. In our experiments we used anti-CD3 and antiCD8 antibodies; (ii) a negatively charged coating that shields the NPs to minimize off-target binding by reducing the surface charge of the NPs. Because it is already widely used in drug delivery platforms, we selected polyglutamic acid (PGA) to accomplish this; (iii) a carrier matrix that condenses and protects the nucleic acids from enzymatic degradation while they are in the endosome, but releases them once the particles are transported into the cytoplasm, thereby enabling transcription of the encoded protein. For this, we used a biodegradable poly(β-amino ester) (PBAE) polymer formulation that has a half-life between 1 and 7 h in aqueous conditions; and (iv) nucleic acids that are encapsulated within the carrier and produce gene editing or transient expression of proteins that can permanently alter the phenotype of the T-cell. mRNA is an ideal platform for transient therapeutic protein expression, because it has no potential for genomic integration and does not require nuclear localization for expression. However, unmodified mRNA can activate intracellular toll-like receptors, limiting protein expression and leading to toxicity27. To improve the stability and reduce the immunogenic potential of the mRNA we deliver, we used synthetic versions that incorporate modified nucleotides. For example, substitution of uridine and cytidine with the engineered bases pseudouridine and 5-methyl-cytidine synergistically blocks identification by innate pattern recognition receptors and increases mRNA translation28. The NPs were manufactured utilizing a two-step, chargedriven self-assembly process. First, the synthetic mRNA was complexed with a positively-charged PBAE polymer, which condenses the mRNA into nano-sized complexes (Supplementary Fig. 1a, b). This step was followed by the addition of 4
antibody-functionalized PGA, which shields the positive charge of the PBAE-mRNA particles and confers lymphocyte-targeting. The resulting mRNA nanocarriers had a size of 109.6 ± SE/26.6 nm and an almost neutral surface charge (1.1 ± SE/5.3 mV zeta potential, Supplementary Fig. 1c). T-cell viability following mRNA nanocarrier transfection. Our goal is to streamline the manufacture of cell-based therapies, so we first tested whether simply adding targeted mRNA nanocarriers to an established culture of human lymphocytes is sufficient to choreograph robust transfection in them. We found that when CD3-targeted NPs carrying mRNA encoding a reporter (enhanced green fluorescent protein, eGFP) are incubated with these cells, they not only bind to them but also stimulate receptor-mediated endocytosis, providing entry for the genes they carry (Fig. 2a). Following a single NP application (NP:T cell ratio = 2 × 104:1), we routinely transfected >80% of these primary T-cells (Fig. 2b, c), with transgene expression observed as early as 5 h post-transfection (Supplementary Fig. 2a–c). Thus, this process is fast and efficient. Importantly, it was not necessary to prepare mRNA NPs freshly for each application—it was possible to lyophilize them before use with no change in properties or efficacy (Supplementary Fig. 2d). We also found that CD3targeted nanoparticles selectively bind T lymphocytes, as their interactions with off-target cells were low (Fig. 2b, c; Supplementary Fig. 3). T cell proliferation did not impair NP-uptake/ transfection, as (i) gene transfer into naive versus effector T-cells was comparable (Supplementary Fig. 4a, b), and (ii) addition of nanoparticles to stimulated T-cells at the peak of their expansion (day 17) yielded similar transfection relative to adding nanoparticles to freshly stimulated cells (day 5; Supplementary Fig. 4c). We next assessed the impact of targeted mRNA−carrying NPs on T-cell expansion. Because malignancies often progress quickly, it is important that engineered T-cells can be expanded to clinically relevant scales equally quickly. One widely used approach to multiply polyclonal lymphocytes is to incubate them with beads that are coated with antibodies against TCR/CD3 and co-stimulatory CD28 receptors. We found that even repeated transfections with CD3-targeted NPs did not interfere with T-cell expansion stimulated by these coated beads (Fig. 2d). These results contrast sharply with T-cell electroporation, which we tested side-by-side. Not only did electroporation add complex handling steps (Fig. 2e), it compromised viability of the lymphocytes (Fig. 2f, g) and reduced their yield by 60-fold (Fig. 2e, right panel). Nanoparticle methods integrate into CAR-T cell manufacture. To test our approach in a clinically relevant application, we incorporated NP-mediated mRNA transfection methods into the manufacture of leukemia-specific 19-41BBζ CAR T-cells (Fig. 3a). CD19-targeted receptors are the most investigated CAR-T cell product today, with nearly 30 ongoing clinical trials internationally29. Our ability to perform genome engineering offers the potential to improve the safety and efficacy of CAR-T-cells. For example, we can inhibit expression of endogenous TCRs to avoid graft-versus-host disease, or selectively delete immune checkpoint genes in these cells to strengthen their anti-cancer activity in the suppressive tumor milieu30, 31. Here, we tested the ability of NPs to deliver gene-editing agents by preparing particles carrying mRNA encoding megaTAL nuclease, which targets the constant region (TRAC) of the TCR alpha gene. Taking advantage of the flexibility offered by our NP formulation methods, we included mRNA for the DNA repair endonuclease TREX2 to improve knockout efficiency32, along with eGFP mRNA so we could track transfection. Control particles were loaded with eGFP
NATURE COMMUNICATIONS | 8: 389
| DOI: 10.1038/s41467-017-00505-8 | www.nature.com/naturecommunications
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00505-8
b
a
Day 0
Day 5 2.8
eGFP (transfection)
T cell stimulation with α-CD3/CD28 beads Treatment with TCRα gene editing or control nanoparticles (NPs) Lentiviral transduction with 19–41BBζ CAR
Day 12 73.6
Control eGFP NPs 22.6
98.4 53.5
97.6
24.6
Gene editing TCRα-eGFP NPs
Incubator 37 °C temperature 30 °C 0
1 2 3 Days in culture
98.7
3.6
70.3
18.3
29.5
CD3 (TCR)
d Size (bp) 650 500 400 300 200
60 40 20
e Control NPs: TCRα NPs: TCRα NPs: total cells SortTCRtotal cells + + + – – –
Lentiviral transduction
TCRα locus
21.8
76.1
5.96
24.5
0.84
1.31
24.7
44.9
Edited locus 71.2
56.6 % Editing
0 NP-transfected T cells (total cells)
19–41BBζ CAR
80 60 40 20
100
10
1
Cytolytic activity
i 2,000
80
1,600
60 40 20 0
0 7 10 Days in culture
Cytokine release
100
20:1 10:1
5:1
Effector:Target ratio
1,200 800 400 0 C42 prostate TM-LCL tumor cells cells
FACS sorted TCR-negative (genome edited), CAR T cells FACS sorted TCR-positive (unedited), CAR T cells
(u
ne TC di R (g te -p en d) os om T it e TC ce ive ed R lls ite ne d) g T ativ ce e lls
0
h % Specific lysis
Lentiviral CAR transduction (%)
100
Proliferation
P = 0.8 (n.s.)
IFN-g (pg/ml)
g
f
TCRα NPs
No NPs Enzyme CD3
80
Fold expansion
TCR knock-out efficiency
c
Fig. 3 Nanoparticles can knockout T cell receptors in CAR-programmed lymphocytes. a Integration of nanoparticle (NP) transfection into normal manufacturing of CAR-T-cells. After stimulation with anti-CD3/CD28-coated beads (day 0), CD8-targeted mRNA NPs were introduced on days 1 and 2, then lentiviral transduction with a vector encoding the leukemia-specific 19-41BBz CAR was performed on day 3. We added either NPs carrying mRNAs encoding megaTAL nuclease plus eGFP, or control particles loaded with eGFP mRNA alone. b Flow cytometry of NP transfection efficiencies (based on eGFP signals) correlated with surface expression levels of TCRs (based on CD3 signals) by T-cells following NP treatments. c Summary plot showing editing efficiency as measured by loss of CD3 surface expression at day 14 (n = 6). d Surveyor assay confirming TCRα chain gene locus disruption. e Flow cytometry of lentiviral transduction in genome-edited versus control T-cells. f Bar graph showing mean viral transductions and SE of three independent experiments conducted in duplicate; n.s., not significant g, h Proliferation and cytolytic activity of TCR+ (FACS sorted TCR-positive, unedited 19-41BBz CAR-T-cells) and TCR- (FACS sorted TCR-negative, genome edited) 19-41BBz CAR-T-cells. To measure proliferation, T-cells were co-cultured on irradiated TM-LCL leukemia cells. Cytolytic assays were performed with CD19-expressing K562 target cells. i T cell IFN-γ release was measured with ELISA 48 h after stimulation on CD19+ TM-LCL leukemia cells or control LNCaP C4-2 prostate adenocarcinoma cells. Data from two experiments run in triplicate are shown
mRNA only. We found that, in contrast to eGFP-transfection (which did not impact TCR expression; Fig. 3b, top row), the addition of TCRα-megaTAL-carrying particles to the T-cell culture efficiently disrupted TCR expression by day 5, an effect that was maintained after loss of the mRNA by day 12 (Fig. 3b, bottom row). Average TCR knockout efficiency was 60.8% ( ± SE/17.7%; Fig. 3c), which corresponds with the percentage of NATURE COMMUNICATIONS | 8: 389
indel frequencies (a measure of targeting efficiency) determined using the Surveyor assay (Fig. 3d). Importantly, the presence of mRNA-carrying NPs did not affect virus-mediated gene transfer of the tumor-specific CAR, as we achieved equal transduction efficiencies with a lentiviral vector encoding 19-41BBζ CAR in NP-transfected and non-transfected T-cells (Fig. 3e, f). Following NP-mediated genome editing and lentiviral transduction,
| DOI: 10.1038/s41467-017-00505-8 | www.nature.com/naturecommunications
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00505-8
b
Relative to mode
Jurkat T
Relative Foxo13A mRNA
a T cells
MFI: 847 6,914
Isotype
MFI: 12,684 17,046
Control NPs (GFP+) Foxo13A-eGFP NPs (GFP+)
1.0
0.5
0.0
Foxo1
0 1 3 5 8 Days post Foxo13A NPs
d
c
Day 1 80
Foxo13A-eGFP NPs (GFP+)
60
60
60
40
40
40
20
20
20
0
0
0 s N Ps
Donor 1 2 3
3A
C on Fo tro xo l N 1 P
s N Ps 3A
3A
C on Fo tro xo l N 1 P Ps lN on
ve
**
Fold change (log2) in Foxo13A NPs –3
C
M
ai
C
N
T
*
C on Fo tro xo l N 1 P
*
80
g
tro
lN
Ps
f
tro
ve
on
C
M
ai
C
N
T
80
s N Ps
CD62L
e
Day 20
Day 8
Control NPs (GFP+) % CD62L+
Relative to mode
CD8+ T cells
–2
–1
0
1
2
3
100
All genes TCM signature
–2
FDR
10
Enrichment P-value: < 0.001
10–4 10–6 10–8
Row min
D 6 C 2L S1 D28 PR 1 KL F2
TCM signature up
C
TCM signature down
Row max
h GSEA for genes differentially expressed in Foxo13A NP treated cells (Day 8) CM signature (up)
CM signature (down) 0.0
Enrichment score
Enrichment score
0.4
–0.35
0.0
Normalized enrichment score: 6.61
Normalized enrichment score: –8.77
FWER P-value:
